Delivery System and Probiotic Composition for Animals and Plants

ABSTRACT

Probiotic compositions that comprise one or more bacteria species in spore form, a thickener to form a stabilized suspension and to preferably act as a prebiotic, one or more acids or salts of acids, and optionally a water activity reducer. A system for delivering probiotic compositions by gravity feed or non-contact pump to a point of consumption by a plant or animal, preferably in conjunction with acidified drinking water, comprising a collapsible container with attachable tubing that prevent contamination of the probiotic composition within the container. Delivery may be actuated in response to a timer, motion detector, fluid level sensor, RFID tag, or other mechanism to periodically or continuously dispense a dosage of probiotic composition directly to the soil surrounding a plant or to the water or feed for an animal. A method for increasing beneficial bacteria in an animal&#39;s GIT comprises adding probiotics to acidified drinking water.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/895,775 filed on Oct. 25, 2013.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to stabilized probiotic compositions, includingsynbiotic compositions and compositions used in conjunction withacidified drinking water, and a system and method for delivering thecompositions to animals and plants.

2. Description of Related Art

Probiotics have been used in farm and agricultural applications for manyyears. A primary use for probiotic formulations is as a feed additive,but other uses include the treatment of housing, animal wound care, pondtreatments, and water treatment. When used as a feed additive, theprobiotic material is typically added directly to the feed (known asdirect-fed microbials or “DFM”) and consumed by the animal. DFM productsare commercially available in a variety of product forms, includingpowder, paste, gel, bolus, and capsules, which may be mixed in feed,top-dressed, given as a paste, or mixed into drinking water or a milkreplacer. Usage doses vary by product, from single dose applications tocontinuous feeding application. Most DFM products must be stored in acool, dry area away from heat, direct sunlight, and high levels ofhumidity to avoid damaging the bacteria or rendering the bacteriaineffective as a probiotic. Some forms of commercially available DFMproducts contain bacterial spores, particularly Bacillus species, whichare considered more stable and may have long shelf lives even underharsh environmental conditions, such as elevated temperatures, drynessand pH extremes. These spores will germinate into vegetative cells andgrow when conditions become favorable. Several Bacillus species areapproved by the Food & Drug Administration and the Association ofAmerican Feed Control Officials (AAFCO) for use in DFM products in theU.S., including B. subtilis, B. licheniformis, B. pumilus, B. coagulans,and B. lentus. Other countries have approved use of other Bacillusspecies as probiotic microorganisms.

Even relatively stable bacterial species used in DFM products may besensitive to certain conditions typically found in application of DFMproducts to livestock. For example, the water activity in the feed canreduce the shelf-life of even the most stable bacterial forms. Varioustechniques are known to help increase shelf life of the bacteria in DFMproducts. Microencapsulation is one known method to increase shelf lifeof probiotic formulas to allow use in DFM products. For example, U.S.Pat. No. 6,254,910 discloses a delivery system for delivering unstableor sensitive ingredients, wherein the unstable or sensitive ingredientis coextruded with other food ingredients so that it is encapsulatedwithin an outer layer of food ingredients, with each layer havingspecific moisture contents. Several probiotic species, includingBacillus coagulans, preferably in spore form, are disclosed in the '910patent as unstable or sensitive ingredients that are suitable forencapsulation to protect the probiotic and increase the probability ofsurvival during processing and in the environmental conditions of use.Other encapsulation technologies including spray drying extrusion,emulsion and phase separation have been used, but with limited successand added expense. A known encapsulated probiotic product, known asBac-In-A-Box, is commercially available from SG Austria(http://www.sqaustria.com/probiotics). Newer microencapsulationtechniques using calcium-alginate gel capsule formation appearpromising, but are still in the development stage and are not yetsuitable for large industrial applications.

Similarly, U.S. Pat. No. 5,501,857 discloses a capsule within a capsulehaving bacterial species, such as one from the genera Lactobacillus orBifidobacterium, in the inner capsule and other beneficial ingredients,such as vitamins, in the outer capsule, with each capsule beingsurrounded by a gelatin shell. The capsule within a capsule structureallows the use of multiple beneficial ingredients that are notcompatible within a single capsule. These capsules have the drawback ofrequiring direct oral administration to the animals. Similar direct oraladministration compositions and methods are disclosed in WO 2010/079104,WO 2012/167882, and U.S. Patent Application Publication No.2013/0017174. However, these formulas are generally complex and must beadministered directly to each animal, which is time consuming and may bedifficult.

It is also known to add probiotic bacteria to animal drinking waterusing a biogenerator to produce the bacteria in a vegetative state. Sucha method is disclosed in U.S. Pat. No. 4,910,024, which describes abiogenerator technology and method for delivering temperature-sensitiveprobiotic bacteria in a live, vegetative condition into a potentiallylarge number of domestic animals as a means of increasing nutrientabsorption efficiency and controlling the proliferation of harmfulmicroorganisms in the digestive tracts of such animals. There areseveral drawbacks to the use of an on-site biogenerator to deliverprobiotics. A primary drawback is the difficulty with maintainingsterility or preventing contamination from external sources of bacteria,yeast, and fungi. Ideally, the water used in the biogenerator must bepathogen free and the device must prevent airborne transmission ofundesirable microorganisms, which may grow in the device and beadministered to the animals. These devices also require power andsufficient water pressure. Additionally, the growth of bacteria in thebiogenerator is temperature dependent and the device would need to betemperature controlled to ensure proper growth of the bacteria. Thenormal ambient temperature and environmental variations (such ashumidity) at sites where the device would typically be used, such as abarn, would result in adverse growth variations and inconsistencieswithout temperature and environmental control. DFM products that delivervegetative state bacteria to the animals' feed or drinking water mightalso be more susceptible to the harsh stomach environment once ingestedby the animals and may not survive to reach the intestinal tract wherethe probiotics are generally most effective. Additionally, the quantityof bacteria must be fed to the animals at the appropriate time and in aproper concentration to be effective, and this can be difficult toachieve with existing probiotic delivery technology.

There are also known systems for delivery of sterile liquids. Forexample, U.S. Pat. No. 5,320,256 discloses a system for delivering asterile liquid comprising a compressible reservoir for storing thesterile liquid, a flexible delivery element extending from the reservoirwith the delivery element having a hollow interior, and a series of shutoff valves to allow delivery and prevent backflow of the liquid or airinto the system. The '256 patent is not specifically related to deliveryof probiotic formulas or delivery of sterile liquids to animals or inagricultural settings. Another example, which is specifically fordelivery of medicines, vitamins, nutrients, and the like to animals, isfound in U.S. Pat. No. 6,723,076. The delivery system disclosed in the'076 patent comprises a sealed, collapsible bag with two flexible tubesattached for filling the bag and administering the solution from the bagto an animal with a syringe gun. Although these systems address some ofthe problems associated with probiotic delivery, they do not addressproblems related to the environmental stability of the probioticcomposition or automated and controlled dosing of the probioticcomposition to a feed or water supply.

It is also known to administer probiotics to animals in spore form. Inaddition to the '910 patent discussed above, U.S. Pat. No. 4,999,193discloses adding spores of bacteria, particularly Bacillus cereus (IP5832 strain), to animal feed or drinking water. Spores are known to beable to withstand high temperatures, making them better suited forincorporation into animal feed during manufacture of the feed, whichtypically involves heat. Although the use of spore form bacteriaaddresses the problems associated with temperature stability, the priorart does not provide an adequate delivery system that eliminates outsidecontamination while achieving controlled delivery in a manner that iseasily used in existing facilities without requiring any retrofitting oradditional power sources.

In addition to providing probiotics, it also known to provide prebioticsin combination with probiotics. This combination is generally known assynbiotics. For example, WO 2012/027214 discloses a synbioticcombination of spore form Bacillus bacteria with prebioticcarbohydrates, including arabinoxylan, arabinoxylan oligosaccharides,xylose, soluble fiber dextrin, soluble corn fiber, and polydextrose. Aprebiotic is a non-digestible carbohydrate or soluble fiber thatprovides a beneficial physiological effect on the host by selectivelystimulating the favorable growth or activity of gut beneficial bacteriaand/or reducing pathogenic populations. Prebiotics are resistant todigestive gastric acid and digestive enzymes in the animal's stomach andsmall intestine and are able to reach the large intestine substantiallyintact or only partially degraded (other than dissolving in waterpresent in the gastrointestinal tract). Once in the large intestine, theprebiotics provide a carbohydrate food source for beneficial bacteriaand undergo complete or partial fermentation in the colon (part of thelarge intestine within which additional nutrient absorption occursthrough the process of fermentation). Fermentation occurs by the actionof bacteria within the colon on the prebiotic food mass, producing gasesand short-chain fatty acids (SCFA). The production of SCFAs, such asbutyric acid, acetic acid, propionic acid, and valeric acids, isincreased when prebiotics are added to animal feed. Scientific studieshave indicated that SCFAs have significant health benefits. Byincreasing beneficial bacterial populations, prebiotics also suppressthe populations of pathogenic bacteria in the colon, such as Clostridia,E. Coli, and Salmonella.

A variety of prebiotics are known, including polysaccharides,oligosaccharides, fructooligosaccharides (FOS), galactooligosaccharides(GOS), soya-oligosaccharides (SOS), xylo-oligosaccharides (XOS),pyrodextrins, isomalto-oligosaccharides (IMO), and lactulose. Specificwater-soluble dietary fiber prebiotics also include Fructans (inulin),Xanthan Gum (E415), Pectin (E440), Natriumalginat (E401), Kaliumalginat(E402), Ammoniumalginat (E403), Calciumalginat (E404), PGA (E405), Agar(E406), and Carrageen (E407). Ingestion of these prebiotic fibers canchange how other nutrients and chemicals are absorbed through bulkingand viscositys and can also change the nature of the contents of thegastrointestinal tract, having been shown to increase populations ofLactobacilli and Bifidobacteria in the intestine and cecum of livestock.In poultry studies (particularly studies regarding broilers), providinga synbiotic combination of probiotic and prebiotic has been shown toincrease the villus/crypt ratio (or ratio of villus height:crypt depth).The villus/crypt ratio is an indicator of the likely digestive capacityof the small intestine. It is within the small intestine that the finalstages of enzymatic digestion occur, liberating small molecules capableof being absorbed, such as, sugars, monosaccharides, disaccharides,amino acids, dipeptides, and lipids. All of this absorption and much ofthe enzymatic digestion takes place on the surface of small intestinalepithelial cells, and to accommodate these processes, a huge mucosalsurface area is required. The villi are minute (finger-like, hair-like,worm-like) projections from epithelial lining of the small intestine.Villus height is measured from the tip (top) of the villus to thevillus-crypt junction. The villi are filled with blood vessels where thecirculating blood takes picks up the nutrients. The crypt is the areabetween villi. Crypt depth is defined as the depth of the invagination,or in folding of the wall, between adjacent villi. Measurements forcrypt depth are measured from the base upwards to the region oftransition between the crypt and villus. Villus surface area iscalculated by using formula, VW/2 times VL, where VW equals the villouswidth and VL equals villus length. More surface area provides moreabsorption of nutrients. The increase in villus/crypt ratio found in thepoultry symbiotic study indicates an increase in digestion andabsorption of nutrients.

Some studies have also shown the importance and benefits of this kind ofsynergy between probiotics and prebiotics and the effectiveness inhelping young animals to achieve better growth performance. Studiesusing B. subtilis as the probiotic and inulin as the prebiotic haveshown that the combination is more effective in swine and poultrypopulations than the use of B. subtilis alone. For example, thissynbiotic combination was shown to reduce (P<0.05) excreta pH,intestinal digesta and cecal content pH compared with a control group.The combination also modulated the ileal and caecal microfloracomposition by decreasing (P<0.05) numbers of Clostridium and Coliformsand increasing (P<0.05) numbers of Bifidobacteria and Lactobacillicompared with a control group. In another study, weaned piglets wereshown to have increased levels of butyrate (a SOFA) when fed with a dietcontaining prebiotics. The importance of butyrate on gut improvement iswell known, as this is crucial to optimize nutrient absorption.

Another issue encountered in animal and plant watering systems isbacteria populations in the drinking water and water transport systems.Municipal water supplies typically have some levels of bacteria presentand local sources of water (such as an on-site pond) may containbacteria from surrounding soil, fish, and run-off. These bacteria areknown to result in or contribute to the formation of biofilms in thedrinking water system. Biofilms are formed when microbial cells attachto surfaces in the water system, such as pipes and drinkingnipples/nozzles, and form a film or slime layer. These biofilms canbuild-up, resulting in clogging parts of the system, or portions of thebiofilm may also break-off causing additional clogging in other areas ofthe system, which reduce the amount of water available to the animals orplants. One known method for removing biofilms in these water systems isto flush the film by increasing the pressure in the water line. Thismethod may cause damage to parts of the water system and typicallyleaves behind a mineral deposit from the biofilm, which will serve as ashelter for micro-organisms and result in the biofilm beingreestablished. Chemical treatment products, such as chlorine andhydrogen peroxide are also known to be used and have good sanitizingabilities; however, these products are not beneficial to gut health forthe animals or soil health for plants and may even be harmful.

It is also know to acidify the water by adding certain organic acids tothe water. The use of acidified water is beneficial for several reasons,including that the acid, in its non-dissociated form, can penetratethrough the bacterial wall and destroy certain microorganisms, which canreduce biofilm formation in the water system, aid in keeping drinkingtrough, nipples/nozzles clean, and can reduce the number of bacteria inthe water. Since the bacteria in the water may be pathogenic and maycause illness when ingested by the animal, reduction of the bacteria inthe water may be particularly helpful in light of bans on antibiotic usein certain geographic areas, such as Europe. Additionally, when ingestedby animals in sufficient quantities to result in a stomach pH belowabout 6, the growth of pathogenic microorganisms (from other sources,such as food) is inhibited. Typically, weak organic acids, such asacetic acid, butyric acid, lactic acid, and sorbic acid, are used toacidify drinking water. There are a number of drawbacks to ordifficulties with acidifying drinking water. For example, it can bedifficult to maintain the pH level at a desired range (below 7 andusually below 5.5). Applying single acids in drinking water typicallyresults in the pH decreasing quickly, which can have negative results,such as less water intake and decreased performance, including lowerfeed conversion rate and lower daily weight gain in the animals. Forexample, during a period of disease, pigs will drop their feed intakebut maintain their water intake. Thus, palatable water is important forthe GIT health of the animals. The use of acids can also be corrosive tometal components in the water system, resulting in added repair andreplacement costs. To decrease these effects, a mix of organic acids maybe used to acidify the drinking water, since the mix has a bufferingeffect that makes the pH decrease slowly. A synergistic mix of organicacids has also a greater antibacterial effect, is more tasteful, and isless corrosive when compared with single acids. Incorrect use ofacidified drinking water can also result in proliferation of bacterialpopulations and growth of algae (which can result in further clogging ofthe water system) and reduction of feed intake (which can result indecreased weight gain and inadequate absorption of nutrients).Additionally, some acids are known to cause fungal growth, which canclog system parts and be detrimental to the animal.

Although it is known to use probiotics alone or in combination withprebiotics and to separately use acidified drinking water to providehealth benefits to animals and to improve cleanliness in plant andanimal water systems, it has not previously been known to combineprobiotics, prebiotics, and acidified drinking water together to providesynergistic health benefits.

SUMMARY OF THE INVENTION

This invention relates to stabilized probiotic compositions, includingcompositions comprising probiotics and prebiotics, and a system andmethod for delivering the compositions to animals and plants, includingdelivering the compositions with acidified water to provide additionalbenefits. The probiotic compositions comprise suspended probiotic sporesthat are stable over a wide range of environmental conditions, includingtemperature fluctuations that would typically be encountered in farm andagricultural settings. The compositions are thermally stable and willnot settle, change composition or activity under the extreme conditionsfound in these animal farming and similar settings. The preferredprobiotic compositions according to the invention comprise one or morespecies from the Bacillus genus in spore form, which are stable duringadverse conditions.

According to one preferred embodiment, the composition comprisesbacteria spores, about 0.00005 to 3.0% by weight surfactant, about 0.002to 5.0% by weight thickener, and optionally about 0.01 to 2.0% by weightof acidifiers, acids, or salts of acids (including those used as apreservative or stabilizer), with the balance being water. According toanother preferred embodiment, the composition comprises bacterialspores, about 0.1 to 5.0% by weight thickener, about 0.05 to 0.5% byweight acids or salts of acids, optionally about 0.1-20% by weight wateractivity reducers, and optionally about 0.1% to 20% additional acidifier(acids or salts of acids), with the balance being water. The optionalacidifiers reduce the pH of the composition to beneficial levels and maybe used to acidify smaller quantities of drinking water when thecomposition is added to drinking water according to a preferred methodof use, as discussed in more detail below.

Most preferably, the bacterial spores in both preferred embodiments arein a dry, powder blend of 40-60% salt (table salt) and 60-40% bacteriaspores that combined make up about 0.1 to 10% by weight of thecomposition. The compositions preferably comprise around 1.0×10⁸ toaround 3.0×10⁸ cfu/ml of the composition (spore suspension), which whendiluted with drinking water (for animal watering applications) providearound 10⁴ to 10⁶ cfu/ml bacterial strains in the drinking water. Mostpreferably, the thickener in both preferred embodiments is one that alsoacts as a prebiotic, such as xanthan gum, to provide additionalbenefits.

The system and method for delivering probiotic compositions, andpreferably those compositions according to the invention and includingprebiotics, or other treatment compositions or sterile liquids comprisespackaging the compositions or liquids in a container and delivering themdirectly to a planter or a water or feed station using gravity feed or abattery powered non-contact pump. The compositions or liquids arepreferably packaged in a container, most preferably a collapsible pouchor bag with an attached or attachable tube (similar to an IV-bag or aMylar bag with an integrated dispensing port attachable to tubing).Depending on the type of container used, the container and tubing may besterilized or may be used without sterilization. By using the systems ofthe invention, the probiotic composition is protected from contaminationby other, outside bacterial species or fungi or the like, while it isbeing stored at the site of consumption prior to being dispensed to theanimal or plant. The composition or liquid is delivered from thecontainer through the delivery tube to animal feed stations, animalwater supply troughs, pressurized drinking water lines, ponds, planters,and the like. The delivery through the tube is controlled by anon-contact pump or gravity feed with a valve to control the flow of thecomposition. The pump or valve are controlled so that the flow of thecomposition can be selectively started and stopped for certain durationsas needed to achieve a proper dosage or volume of discharge, dependingon the particular composition or liquid involved and the animal or plantspecies to which the composition or liquid are being delivered, or totime the dosage to match a particular feeding and drinking schedule. Thepump or valve may also be controlled in response to external stimuli,such as motion or light. It is not necessary to vent the collapsiblebag, so airborne contaminates and undesirable bacteria are notintroduced into the feed container. According to one preferredembodiment, a duck-bill type valve or similar mechanism is attached tothe end of the delivery tube to prevent any contaminants or undesirablebacteria from growing in the end of the tube. Whether used with anon-contact pump or gravity feed, the feed container is preferably hungat a sufficient height above the pump or discharge point to providesufficient hydrostatic pressure to feed the pump or discharge thecomposition, and to protect it from the animals. To aid in securing thecontainer and protecting it from possible puncturing, it may optionallybe placed or hung inside a protective cabinet or housing. The system andmethod for delivering compositions and liquids according to theinvention is simple and low-cost and does not require an on-site sourceof sterile water or electric power supply to operate.

BRIEF DESCRIPTION OF THE DRAWINGS

The system and method of the invention are further described andexplained in relation to the following drawings wherein:

FIG. 1 is a side elevation view of one embodiment of a delivery systemaccording to the invention;

FIG. 2 is a side elevation view of another embodiment of a deliverysystem according to the invention;

FIG. 3 is a side elevation view of an alternative container and tubingfor use with the delivery systems according to the invention;

FIG. 4 is a front perspective view of another preferred embodiment of adelivery system according to the invention;

FIG. 5 is a rear perspective view of the embodiment of the deliverysystem of FIG. 4;

FIG. 6 is a partial front perspective view of the delivery system ofFIG. 4 within a support housing; and

FIG. 7 is a side elevation cross-sectional view of the delivery systemof FIG. 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A probiotic composition according to one preferred embodiment of theinvention comprises one or more bacterial species, a surfactant, and athickener, and optionally one or more acidifiers, acids or salts oracids to act as a preservative. A probiotic composition according toanother preferred embodiment of the invention comprises one or morebacterial species, a thickener, one or more acidifiers, acids or saltsof acids, and optionally a surfactant. Either embodiment may alsooptionally include prebiotics, to the extent the thickener is not also aprebiotic or in addition to any thickener that is a prebiotic. Eitherembodiment may also optionally include one or more water activityreducers. Most preferably, the compositions according to the inventioncomprise various species of suspended probiotic spores, as described inmore detail below. The use of these species in spore form increases thestability of the probiotics in the harsh environmental conditions,particularly temperature fluctuations that occur in stables, barns, andother farm and agricultural settings.

A suitable thickener is included in the composition according to bothpreferred embodiments. The thickener is preferably one that does notseparate or degrade at varying temperatures typically found innon-climate controlled environments, such as barns, farms, andnurseries. The thickener aids in stabilizing the suspension so thebacterial mixture remains homogenous and dispersed through a volume ofthe composition and does not settle out of the suspension. When usedwith the system and method of delivery described below, this ensuresthat the concentration of probiotic materials is evenly distributedthroughout the container so that the dosage of probiotic materialdelivered remains consistent or relatively consistent (depending on thespecific delivery method and control mechanism used) throughout atreatment cycle.

The most preferred thickener in either embodiment is xanthan gum, whichis a polysaccharide composed of pentasaccharide repeat units of glucose,mannose, and glurcuronic acid and a known prebiotic. Unlike some othergums, xanthan gum is very stable under a wide range of temperatures andpH. Xanthan gum, like all soluble fibers, helps balance intestinal pHand tends to slow the movement of food and extends the mouth to cecumtransition time. This slowing may allow more time for the spores togerminate in the stomach before reaching the intestines, which allowsfor use of the more stable spore form bacteria rather than the use ofvegetative bacteria that may not survive the harsh environmentalconditions of use or may not survive the animal's stomach. Anotherpreferred thickener is acacia gum, which is also a known prebiotic.Other preferred thickeners include locust bean gum, guar gum and gumarabic, which are also believed to be prebiotics. In addition toprebiotic benefits, these fibers do not bind to minerals and vitamins,and therefore, do not restrict or interfere with their absorption andmay even improve absorption of certain minerals, such as calcium. Otherthickeners that are not considered prebiotics may also be used.

Either embodiment may optionally include one or more prebiotics, whichare preferably used if the thickener used is not a prebiotic but mayalso be used in addition to a prebiotic thickener. Prebiotics areclassified as disaccharides, oligosaccharides and polysaccharides, andcan include Inulin, Oligofructose, Fructo-oligosaccharides (FOS),Galacto-oligosaccharide (GOS), trans-Glacto-Oligosaccharides (TOS) andShort-Chain Fructo-oligosaccharides (scFOS), soy Fructo-oligosaccharide(soyFOS), Gluco-oligosaccharides, Glyco-oligosaccharides, Lactitol,Malto-oligosaccharides, Xylo-oligosaccharides, Stachyose, Lactulose,Raffinose. Mannan-oligosaccharide (MOS) are prebiotics may not enrichprobiotic bacterial populations, but will bind with and remove pathogensfrom the intestinal tract and are believed to stimulate the immunesystem.

Both embodiments preferably include one or more acidifiers, acids, orsalts of acids to act as a preservative or to acidify the composition.Preferred preservatives are acetic acid, citric acid, fumaric acid,propionic acid, sodium propionate, calcium propionate, formic acid,sodium formate, benzoic acid, sodium benzoate, sorbic acid, potassiumsorbate, and calcium sorbate. Other known preservatives, preferablygenerally regarded as safe (GRAS) food preservatives, may also be used.One or more of these same acids or salts or acids may also be optionallyadded as an acidifier, in addition to any amount used as a preservative.Depending on the dosing mechanism and environment, the optionalacidifier may be used to acidify a smaller amount of drinking water,such as the water at a single, smaller scale trough. For larger watersystems and multiple troughs or drinking stations, it is preferred touse a separate acidification system since larger quantities of acid orsalts or acids will be need to reduce the pH of the larger volume ofwater. Even if not used to fully acidify the drinking water, these acidsand salts of acids aid in reducing the pH of the composition.Preferably, the pH of the composition is between about 4.0 and 7.0. Morepreferably it is between about 4.0 and 5.5 and most preferably around4.5. Reducing the pH of the composition may have antimicrobial activitywith respect to yeast, molds, and pathogenic bacteria.

One or more water activity reducers, such as sodium chloride, potassiumchloride, or corn syrup (a 70% solution of corn syrup), are optionallyincluded in the composition according either preferred embodiment. Thewater activity reducer aids in inhibiting microorganism growth, so thatthe bacterial spores do not prematurely germinate while the compositionis being stored prior to the time it is discharged to the point ofconsumption by the animals or plants to be treated. They also aid ininhibiting growth of contamination microorganisms

The first embodiment preferably includes a surfactant, but it isoptional in the second embodiment. The surfactant is preferably one thatis safe for ingestion by animals, although other surfactants may be usedwith other applications, such as delivery to plants. Most preferably,the surfactant is Polysorbate 80. Although any GRAS or AAFCO approvedsurfactants or emulsifiers may be used with either embodiment, there areconcerns that some animals may not tolerate all approved surfactantswell. Because the benefits of the surfactant in stabilizing thesuspension so the bacterial mixture remains homogenous and does notsettle out may also be achieved by the use of the thickener, it is notnecessary to add the surfactant. If a surfactant is used in thecomposition according to this second embodiment, it is preferably usedin about the same weight percentage range as in the first embodiment.

Most preferably, the bacterial species used in both embodiments are oneor more species from the Bacillus genus. The most preferred species forthe probiotic bacteria include the following: Bacillus pumilus, Bacilluslicheniformis, Bacillus amylophilus, Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus clausii, Bacillus firmus, Bacillusmegaterium, Bacillus mesentericus, Bacillus subtilis var. natto, orBacillus toyonensis, but any Bacillus species approved as a probiotic inthe country of use may also be used. It is preferred that the bacteriaare in spore form, as the spore form is more stable to environmentalfluctuations, such as ambient temperature changes. Additionally, ascompared to vegetative state DFM, spores are believed to be better ableto survive through the stomach once ingested by an animal to germinatein the intestines, where they are beneficial. Most preferably, thespores used in the compositions according to the invention are a drypowder blend that comprises around 40-60% salt (table salt) and 60-40%bacterial spores. The spores are spray-dried from a liquid fermentationconcentrate. Salt is used to dilute the pure spray-dried spore powder toa standard spore count in the final spore powder blend. Duringproduction fermentation, different Bacillus strains will grow atdifferent rates, resulting in varying final count numbers for thefermentation batch liquor. The fermentation liquor is centrifuged toconcentrate the spores in the liquor. Then, the concentrated liquor isspray-dried which results in a powder containing only Bacillus spores.The addition of salt to the spray-dried Bacillus spore powder aids instandardizing the spore blend count per gram from batch to batch. Otherforms of bacterial spores or spore blends may also be used. Mostpreferably, the dry spore blend is pre-mixed with a portion of the waterused in the composition, around 3-30% of the total water, and theresulting bacteria spore solution is added to the other ingredients,including the remaining water. This aids in dispersing the bacteriaspores throughout the composition.

A probiotic composition according to a first preferred embodiment of theinvention preferably comprises bacterial spores that provide 10⁸ cfu/mlof the spore suspension (most preferably around 1.0×10⁸ to around3.0×10⁸ cfu/ml of composition, which, when diluted in drinking waterprovides approximately 10⁴ to 10⁶ cfu/ml drinking water), 0.00005 to3.0% surfactant, and 0.002 to 5.0% thickener, and optionally the about0.01 to 2.0% of one or more acids or salts of acids as a preservative. Aprobiotic composition according to a second preferred embodiment of theinvention comprises bacterial spores that provide 10⁸ cfu/ml of thespore suspension (which, when diluted in drinking water providesapproximately 10⁴ to 10⁶ cfu/ml drinking water), about 0.1 to 5.0%thickener (preferably one that also acts as a prebiotic), about0.05-0.5% of one or more preservatives, optionally about 0.1-20% of oneor more water activity reducers, and optionally 0.1-20% of one or moreacidifiers. The balance of the composition in both preferred embodimentsis water and the percentages herein are by weight. It is preferred touse deionized or distilled water, to remove salts or outside bacteria,but tap water or other sources of water may also be used.

Several examples of probiotic compositions according preferredembodiments of the invention were made and tested for differentparameters. These compositions are set forth in Table 1 below.

TABLE 1 Formula No. Ingredient 1 2 3 4 5 6 7 8 Potassium Sorbate 0.33%0.33% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% Citric Acid 0.34% 0.34% 0.1% 0.1%5.0% 0.1% 0.1% 0.1% Sodium Benzoate 0.33% 0.33% 0.1% 0.1% 0.1% 0.1% 0.1%0.1% Benzoic Acid — — — 0.1% — 0.1% 0.1% — Sorbic Acid — — — 0.1% — —0.1% — Sodium Propionate — — — — 10.0%  0.1% — — Xanthan Gum  0.2%  0.2%0.2% 0.3% 0.4% 0.4% 0.5% 0.5% Sodium Chloride  0.2%  0.2% — 0.2% — 0.2%0.1% 0.2% Potassium Chloride — — — — — — 0.1% 0.1% Spore Blend  0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1%

The balance of each composition is water (around 1 L in these samples).Deionized water was used in each composition, except composition No. 1,which used tap water. The percentages indicated are by weight. Eachformula was targeted to have a pH between about 4.0 and 5.5, but someformulas were found to have actual pH values far less than expected.Formula No. 1 was targeted to have a pH between 5.0 and 5.5, but itsactual pH was around 2.1-2.3, which is too low and may be harmful to thespores, create stability issues with packaging, and be subject to morerestrictive transportation regulations. Formula No. 1 also exhibitedweak thickening. Formula No. 2 is the same as No. 1, except the sourceof water is different. Formula No. 2 had an actual pH of around 2.2-2.3and also exhibited weak thickening. The amount of acids and salts ofacids in Formula No. 3 was decreased to raise the pH and to determine ifthe thickness improved while using the same amount of thickener as inNos. 1 and 2. While Formula No. 3 was an improvement over Nos. 1 and 2,it still exhibited weak thickening and its actual pH was 6.6, over thetarget value range. Additional acids were added to Formula No. 4 tolower the pH and additional thickener was added. Formula No. 4 hadimproved thickening, but further improvements in thickening would bebeneficial. The amount of acid in Formula No. 5 was substantiallyincreased, which resulted in an actual pH of around 1.0. The amount ofacid in Formula No. 6 was decreased and the thickener increased, whichresulted in a composition that was too thick to drop. Formula No. 7increased the thickener and amount of water activity reducers, butexhibited issues with mixing of benzoic acid and sorbic acid. Thebenzoic acid and sorbic acid were removed in Formula No. 8. Formula Nos.1-7 provided 2×10¹¹ cfu/gm and No. 8 provided 1×10¹¹ cfu/gm bacteriaspores. Of these sample formulas, No. 8 is the most preferred as itexhibited adequate thickening and had an actual pH of around 4.5+/−0.2,and used less spore blend.

It is preferred that the compositions according the embodiments of theinvention use around 0.01% to around 0.3% bacteria spore blend and morepreferably between about 0.03% to 0.1% bacteria spore blend. A reductionin the amount of spore blend used substantially reduces the costs of thecomposition. Depending on the end use application, differing amounts ofspore blend may be used in the compositions according to the invention.For example, smaller percentages of spore blend may be used in thecompositions for use with chickens, whereas larger percentages would beused in composition for use with pigs.

A composition according to formula No. 8 was tested for shelf-life atvarious temperatures. Samples of Formula No. 8 were sealed in a plasticbag, such as one used in a preferred delivery system as described below,and stored for two months at temperatures around 4-8° C. (39-46° F.),30° C. (86° F.), and 35° C. (95° F.) to simulate typical temperatureranges in which the probiotic composition may be stored and used inagricultural settings. At the end of the first month of the storageperiod, each sample was observed and tested. All three samples had a pHof around 4.5 and there was no settling, layering or change ofappearance in any of the three samples, indicating that the bacteriaspores remained suspended and dispersed throughout the compositionduring the storage period. None of the samples contained any fungalcontamination or gram-negative bacteria contamination. At time countzero (when the samples were initially stored), each sample containedbacteria spores of around 2.12×10⁸ cfu/mL. At the end of the one monthstorage period, the samples contained bacteria spores of around 2.09×10⁸cfu/mL spore suspension (lowest temperature sample), 1.99×10⁸ cfu/mL(middle temperature sample), and 2.15×10⁸ cfu/mL (high temperaturesample). The bacteria counts are somewhat variable in different samples,especially thickened samples; however, these are considered to becomparable counts. Each sample was tested again after two months instorage. The samples contained bacteria spores of around 2.08×10⁸ cfu/ml(lowest temperature sample); 2.01×10⁸ cfu/ml (middle temperaturesample); and 2.0×10⁸ cfu/ml (high temperature sample). The target shelflife is around 2×10⁸ cfu/ml spore suspension, so the samples are withinthe targeted shelf life after two months of storage. These test resultsdemonstrate that probiotic compositions according to a preferredembodiment of the invention are stable over a range of temperatures,with the bacteria spores remaining viable, suspended, and dispersedthroughout the composition. The spore blend (40-60% spore powder and60-40% salt) used in each sample formula was the same, providing atleast around 2×10¹¹ spores/gram. The spore species in the blend weremultiple Bacillus subtilis and Bacillus licheniformis strains. The sporeblend powder was premixed with 100 mL of water with stirring for 30minutes prior to adding to the other ingredients. Premixing with wateraids in mixing the spore blend with the other ingredients and dispersingthe spores throughout the composition.

Another aspect of the invention is a system and method for deliveringprobiotic compositions, and preferably probiotic compositions accordingto the invention as described herein, directly into animal feed ordrinking water at the point of consumption. Although it is preferred touse probiotic compositions comprising one or more Bacillus species asaccording to the compositions of the invention, the system of theinvention may be used with compositions comprising other bacteria generaand other species. For example, one or more species from the followinggenera: Bacillus, Bacteroides, Bifidobacterium, Pediococcus,Enterococcus, Lactobacillus, and Propionibacterium (including Bacilluspumilus, Bacillus licheniformis, Bacillus amylophilus, Bacillussubtilis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus firmus,Bacillus megaterium, Bacillus mesentericus, Bacillus subtilis var.natto, or Bacillus toyonensis Bacteroides ruminocola, Bacteroidesruminocola, Bacteriodes suis, Bifidobacterium adolescentis,Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacteriuminfantis, Bifidobacterium longum, Bifidobacterium thermophilum,Pediococcus acidilacticii, Pediococcus cerevisiae, Pediococcuspentosaceus, Enterococcus cremoris, Enterococcus diacetylactis,Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis,Enterococcus thermophilus, Lactobacillus delbruekii, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillusbuchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillusfarciminis, Lactobacillus cellobiosus, Lactobacillus curvatus,Propionibacterium acidipropionici, Propionibacterium freudenreichii,Propionibacterium shermanii) and/or one or more of the followingspecies: Leuconostoc mesenteroides, Megasphaera elsdennii may be usedwith the system and method of the invention.

Referring to FIG. 1, one preferred embodiment of a delivery system 10 isdepicted. Delivery system 10 preferably comprises a container 12, tubing15, non-contact pump 20, and controller 22. Container 12 is preferably acollapsible bag, similar to an IV bag, containing the probioticcomposition. An upper end 14 of container 12 is preferably a sealed-offsection of the collapsible bag through which one or more holes 26 areprovided to facilitate hanging container 12 from hooks or a nail at thesite of consumption. Alternatively, container 12 may be hung from clipsor similar mechanisms and upper end 14 may include externally protrudingridges to aid in securing container 12 to such clips. Container 12 maybe sterilized, preferably by UV sterilization, prior to container 12being filled with a probiotic composition to ensure there is nocontamination. Optionally, air may be removed from the head space in thecontainer, but it is not necessary. Attached at or attachable to a lowerend of container 12 is a tube 15 having a first portion 16 disposed nearcontainer 12 and a second portion 18 disposed near the point ofconsumption where the probiotic formula will be dispensed. Tube 15 maybe integrally formed with container 12 or pre-attached to container 12,as will be understood by those of ordinary skill in the art, prior toshipping to the site of use. Tube 15 may also be sterilized, preferablyby UV sterilization, prior to shipping. When integrally formed with orpre-attached to container 12, the distal end of tube portion 18 ispreferably sealed with a removable seal or covering, or may include avalve, to seal-off container 12 once filled with a probioticcomposition. The seal or covering would be removed or the valve openedwhen the probiotic composition is ready to be dispensed at the point ofuse.

Tube 15 passes through a non-contact pump 20, such as a peristalticpump, which allows the probiotic composition to be pumped from container12 through tubing 15 to be dispensed at the point of consumption withoutany potentially contaminating contact with the pump or the exteriorenvironment in which system 10 is being used. Container 12 preferablycollapses as it is emptied by the pumping action without the need for anair vent. Pump 20 is preferably battery operated so that an externalpower source is not needed, making it easy to add system 10 to existinganimal feed or drinking locations, but may also be adapted to connect toan outlet or other external power source.

Container 12 is preferably hung above pump 20 at a sufficient height toprovide the head pressure needed to deliver the probiotic composition toan inlet on pump 20. Most preferably, the lower end of container 12 willbe hung at least 6 inches above the inlet to pump 20. The distal end oftube portion 18 preferably includes a “duck bill” valve or similarmechanism to prevent any contamination by backflow into the tubing. Thedistal end of tube portion 18 may also include spray or dispersionstructures designed to dispense the probiotic composition in a widerpattern. A wider dispersion pattern is particularly preferred if theprobiotic composition is dispensed into stagnant drinking water, a largewater or feed trough, dry animal feed, or animal housing or beddingmaterial in order to spread the composition over more surface area. Awider dispersion pattern may also avoid saturating dry feed withmoisture. The distal end of tube portion 18 may also be split intomultiple tubes or attached to a manifold to facilitate delivery of theprobiotic composition to multiple locations, such as multiple drinkingtroughs within a barn.

A controller 22 is preferably connected to pump 20 to periodicallyactivate the pumping action. Controller 22 may comprise one or morecontrol mechanisms for activating pump 20, such as a simple timer thatactivates pump 20 for a given duration or cycle at specified timeintervals. For example, controller 22 may be a timer programmed toactivate pump 20 for a 60 second cycle every six hours. Other controlmechanisms may also be used in addition to or in place of a timer. Forexample, a motion detector may be used in conjunction with a timer toactivate pump 20 for a 60 second cycle when motion is detected for aspecified period of time. Such a mechanism would allow the probioticcomposition to be delivered to the animal's drinking water or feed whenthe presence of animals is detected by motion. This has the advantageover a timer only control mechanism, which may dispense the probioticcomposition when no animals are present and may result in wasting theprobiotic. The bacteria in the probiotic compositions according to theinvention may survive for hours after being dispensed, so that they arelikely to still be viable when an animal arrives to feed or drink evenif it was dispensed when no animals are present, so dispensing whenanimals are present is not critical. The controller 22 may also beconfigured to sense ambient light conditions (such as daylight orsimulated daylight, when animals are more likely to be eating ordrinking) or temperatures, or other variables that may be encountered atthe point of consumption. Sensing these conditions may result in theprobiotic composition being dispensed at times when the animals are morelikely to be active and feeding or drinking to avoid wasting theprobiotic composition.

The controller 22 may also be configured to sense RFID tags, or similartechnology, attached to the animals. The controller 22 would read thesignal from the RFID tag, determine which animal is present, andselectively trigger the pump to dispense the probiotic composition ifthe animal is to receive the probiotics. This may be particularly usefulif it is desired to provide probiotics to certain animals, but is notconsidered necessary for other animals, without requiring individualadministration of a capsule or injection. The use of RFID tags may alsoallow the system to monitor how long an animal is present at the wateror feeding station, which may be used to correlate how much probioticwas actually ingested. Other control mechanisms may be used with theinvention, as will be understood by those of ordinary skill in the art.Controller 22 is preferably battery operated so that an external powersource is not needed, making it easy to add system 10 to existing animalfeed or drinking locations, but may also be adapted to connect to anoutlet or other external power source. Controller 22 may be incorporatedinto pump 20 and need not be a separate component of system 10.

Referring to FIG. 2, a preferred embodiment of a delivery system 110 isdepicted. Delivery system 110 relies on gravity to dispense theprobiotic compositions, rather than a pumping system. Delivery system110 preferably comprises a container 112, tubing 115, valve 124, andcontroller 122. Container 112 is preferably a collapsible bag, similarto container 12. Tubing 115 preferably comprises a first portion 116disposed near container 112 and a second portion 118 disposed near thepoint of consumption where the probiotic composition will be dispensed.Rather than passing through a non-contact pump, as in system 10, a valve124 is disposed along the length of tubing 115 (separating portion 116from 118). The features previously described for container 12 and tubing15 are also applicable to container 112 and tubing 115. Valve 124 ispreferably a pinch valve, but other types of valves may also be used.Actuation′ of valve 124 from an open to closed position is controlled bycontroller 122. The various control mechanisms for controller 122 arethe same as for controller 22. As the probiotic composition in container112 is dispensed, the head pressure will change and the amount ofprobiotic dispensed will vary over time unless the controller 122 isprogrammed to keep the valve open longer or to open with greaterfrequency as the volume of probiotic composition within container 112decreases. For simplicity, controller 122 may be programmed for thelowest expected head pressure. Variations in the programming may be setaccording to the height at which container 112 is hung. Most preferably,the lower end of container 112 will be hung at least 6 inches above thelevel of dispensing at the end of tubing portion 118. Controller 122 ispreferably battery operated so that an external power source is notneeded, making it easy to add system 110 to existing animal feed ordrinking locations, but may also be adapted to connect to an outlet orother external power source.

As shown in FIG. 3, an alternate configuration for a container andtubing suitable for use with system 10, 110, or 210 (as discussed below)is shown. Container 212 comprises an integrally formed or pre-attachedspout 228. Spout 228 is preferably sealed with a removable cap duringshipping and storage. Spout 228 preferably has threads that mate withthreads inside the cap so that it is easily removed when the probioticcomposition inside container 212 is ready to be dispensed.Alternatively, spout 228 may have a removable seal or covering toseal-off container 212, without requiring the use of a cap. Tubing 215preferably comprises an upper end 216 that comprises a connector 232designed to mate with spout 228 to allow fluid communication betweencontainer 212 and tubing 215 through spout 228. Preferably, connector232 comprises threads that mate with threads on spout 228. Prior toshipping, it is preferred that tubing 215 be sterilized, preferably byUV sterilization, and that both ends of tubing 215 be sealed to preventcontamination during shipping and storage. The seals would be removedwhen tubing 215 is attached to container 212 and ready to dispense theprobiotic composition. Container 212 and tubing 215 may be used with theother parts of delivery systems 10, 110, or 210, depending on whether apump-fed application or gravity-fed application is desired. The featurespreviously described for tubing 15, such as a duck bill valve or spraynozzle, may also be used with tubing 215. Container 212 also preferablycomprises an upper end 214 that forms a sealed-off section through whichone or more holes 226 are provided to facilitate hanging container 212from hooks or a nail at the site of consumption. Alternatively,container 212 may be hung from clips or similar mechanisms and upper end214 may include externally protruding ridges to aid in securingcontainer 212 to such clips.

As shown in FIGS. 4-7, another preferred embodiment of a delivery system210 according to the invention is shown. Delivery system 210 is similarto delivery system 110 in that it relies on gravity to dispense theprobiotic compositions or other fluids, rather than a pumping system.Delivery system 210 has the added benefit of allowing adjustment in thedosage volume using a piston-type valve actuated by a simple motor tovary the volume of dosing based on how long the motor is operated.Delivery system 210 preferably comprises a container 212, a meteringvalve 234, a valve mounting structure 240, a motor 252, a cam 254, and amotor mounting structure 264. Container 212 is similar to that shown inFIG. 3, but other containers and mechanisms for attaching the containerused with system 210 to metering valve 234 and delivery tubing (similarto tubing 216) may be used. Container 212 is preferably hung at anelevated location, as discussed with respect to container 112 of system110. Container 212 comprises a spout 228 that is connectable in fluidcommunication with metering valve 234. Metering valve 234 is preferablya capacity dosage piston system comprising an upper portion 236, a lowerportion 235, and a piston 224. Upper portion 236 attaches to spout 228to form a fluid tight seal. Upper portion 236 may comprise threads tomate with threads on spout 228 to connect the two parts together. Theflow of fluid from container 212 is regulated by the piston 224, whichis actuated by motor 252 and cam 254, as described below. Each pumpstroke of piston 224 causes an amount of fluid from container 212 to bedispensed. Lower portion 235 is preferably configured to allow piston224 to be inserted into the body of lower portion 235 and for slidingmotion of piston 224 in and out of lower portion 235 as piston 224 isactuated by cam 254 to dispense an amount of fluid.

Valve mounting structure 240 preferably comprises a valve mounting base242, a valve slider 246, and a piston bracket 248. Base 242 preferablycomprises one or more slots 244 into which one or more correspondingprotrusions 247 on valve slider 246 are inserted to attach valve slider246 to base 244 in a slidable configuration. Piston bracket 248 ispreferably connected to valve sider 246 and configured to mate with aportion of the body of piston 224, such that when valve slider 246slides up or down relative to base 242, piston 224 correspondinglyslides up or down relative to lower portion 235. The sliding motion ofvalve slider 246 is actuated by cam 254. Cam 254 preferably comprises arotational body 256 and a shaft 258 disposed between and connected torotational body 256 and first shaft connector 262 at one end and to asecond shaft connector 260 at the other end. First shaft connector 262allows shaft 258 to pivot as rotational body 256 rotates. Second shaftconnector 260 connects cam shaft 258 to valve slider 246 through anelongated aperture 250 in valve mounting base 242. Cam 254 is connectedto motor 252 by drive shaft 253. Motor 252 and drive shaft 253 driverotational body 256 to rotate, which is translated into linear movementof valve slider 246 along the length of aperture 250 through cam shaft258 and second shaft connector 260. As valve slider 246 moves up anddown relative to mounting base 242, valve bracket 248 and consequentlypiston 224 are also moved up and down, which actuates opening or closingvalve 234 to start or stop the flow of probiotic composition or otherfluid from container 212. Tubing (similar to tubing 215, which mayinclude all the features previously described for tubing 15) to carrythe probiotic composition or other fluid from container 212 to the pointof delivery, such as a water trough, is disposed through opening 249 inbracket 248, to allow fluid communication through valve 234 when valve234 is in an open position.

Motor 252 is preferably a simple DC gear motor with an internal timingmechanism, but other types of motors may also be used. The timingmechanism activates the motor 252 to move piston 224 to open valve 234for a predetermined amount of time and then activates the motor 252again to close valve 234. Alternatively, the motor may run continuouslyfor a period of time, repeatedly opening and closing the valve 234 untilthe desired dosage of fluid from container 212 is dispensed, thenshut-off until the next predetermined cycle time. The cycle of openingand closing valve 234 would be periodically repeated, such as once every24 hours, once every 8 hours, or any other selected cycle intervalneeded to dose the desired amount of probiotics or other fluid fromcontainer 212. Most preferably, the timing mechanism may be adjustableby a user or include multiple cycle timing options that may be selectedby a user to achieve the desired activation and dosing schedule. Motor252 may also be separately connected to a controller, similar tocontroller 122, and the various control mechanisms are the same as forcontroller 22 or 122. Other types of valves, such as a solenoid valvecontrolled by a programmable timer, may also be used with systemsaccording to the invention to meter a dosage of fluid at given cycles toachieve a desired dosing rate.

Motor mounting structure 264 supports motor 252 and allows it to besecurely attached to any suitable structure in the area where the fluidis to be discharged. One or more apertures 266 are preferably disposedthrough mounting structure 264 to allow it to be secured by screws orany conventional attachment mechanism. Most preferably, system 210 isdisposed in a housing 270 and mounting structure 264 would be secured toan interior bottom wall of housing 270. Housing 270 is partially shownin FIG. 6. Housing 270 preferably comprises walls 272 on the sides,front, back, top and bottom surfaces, but the front, bottom and topwalls are not shown in FIG. 6. Preferably either the front or top wallis a removable or openable door or cover to allow access to the interiorof housing 270 to allow replacement of container 212 and other parts ofthe system 210 as needed. Housing 270 preferably comprises a pluralityof support tabs 274 that extend inwardly from one or more walls 272,preferably from side walls 272, to aid in supporting and securingcontainer 212 within housing 270. A spout retainer 276 is alsopreferably provided to allow insertion of spout 228 and to aid insupporting container 212 within housing. Hooks or clips may also bedisposed on a top wall or near an upper end of a back wall of housing270 to hang container 212 within housing 270 using holes 226 or byclipping onto upper end 214 of container 212. Housing 270 protectscontainer 212 from inadvertent puncturing and could provide someprotection for the motor 252 (or other type of controller) from theenvironmental conditions in the place of use. Most preferably, thecontainers and controllers used with system 10 and 110 are alsopreferably located within a cabinet or housing, such as housing 270shown in FIG. 6, to provide additional protection for these components.Housing 270 or such other cabinet would preferably be located in anelevated position (particularly for systems 110 and 210, which rely ongravity feed) and securely attached to a wall or similar structure.Other parts of the systems 10, 110, or 210 may also be placed withinhousing 270 or such other cabinet.

Containers 12, 112, and 212 are designed to be discarded and replacedwith new containers 12, 112, or 212 when the probiotic contents are allor substantially all consumed over the course of repeated dosing cycles.Preferably tubing 15 and 115 are pre-attached to the containers orintegrally formed with the containers and are similarly discarded at theend of a container cycle. Tubing 215 is also preferably discarded andnew tubing 215 used with each new container 212, but tubing 215 may bereused with a new container if desired. The size and volume of the feedcontainer containing the probiotic composition, treatment composition orother sterile liquid may be scaled according to the use environment.Typically, containers 12, 112, or 212 will be sized to hold 1 liter to25 liters of probiotic composition, treatment composition or othersterile liquid. Although dosing amounts may vary, depending onenvironmental conditions, type of feed mechanism used, and the type andnumber of animals involved, a one liter supply of probiotic compositionaccording to the invention added to drinking water will be sufficient toprovide an average pig with 5.4×10⁹ spores per day for 30 days or couldsupply 2,000 chickens at a rate of 10⁶ spores per day per chicken for 50days. This allows the use of smaller sized containers in mostapplications, which are easier to handle by a single person, but mayrequire more frequent replacement with new containers to replenish thesupply of probiotic composition. Larger sized containers may also beused and containers 12, 112, or 212 may be placed within a cabinet orother housing (such as housing 270 shown in FIG. 6) to help support thesize and weight of the container or may be replaced within largerbottles or barrels as needed.

Systems 10, 110, and 210 may be used to dispense probiotic and/orsynbiotic compositions to animal feed, drinking water, bedding orhousing areas. When dispensed to bedding and housing areas, theprobiotic composition spores might compete with pathogenic bacteria, andshould degrade organic matter, thus reducing odors. Most preferably,systems 10, 110, and 210 are used to dispense probiotic and/or synbioticcompositions to animal drinking water. Frequently, the drinking water isdispensed in a trough with flowing water. The dispensing point at theend of tubing portion 18 or 118 or similar end of tubing 215 may belocated at the head of the trough so that the probiotic composition mayflow downstream to reach multiple animal drinking locations. Othercomponents may be added to these systems which enhance the ability tocontrol the feed of the probiotics. For example, a flow meter toproportion the probiotic feed rate to the water flow rate or a venturito pull the probiotic composition proportional to the water flow ratemay be used. When used with an individual, non-flowing water station, awater level sensor could be incorporated into the system. In combinationwith the controller, the water sensor could track the animal's waterintake in order to determine the amount of probiotic ingested. Thisinformation could be used as part of an optimization program for animalhusbandry. This type of system is also useful with household pets, whichtypically have individual water bowls. Those of ordinary skill in theart will understand the modifications need to incorporate such features.

With respect to system 10, the amount or rate of probiotic compositiondispensed or fed will be a function of the rate of pumping, the durationof the pumping cycle, and the size of tubing used. With respect tosystem 110, the amount or rate of probiotic dispensed or fed will be afunction of the duration the valve is opened, the head pressure, and thesize of tubing used. With respect to system 210, the amount or rate ofprobiotic dispensed or fed will be a function of the number of pumpstrokes or the duration the valve is opened, the head pressure, and sizeof tubing used. The viscosity of the probiotic composition may alsoimpact the amount or rate with which the composition is dispensed. Thedesirable doses of the probiotic compositions will vary depending on theprobiotic used and the particular animal or plant species involved. Forexample, larger size or “finishing” pigs are generally regarded asrequiring some of the highest doses of probiotics to be beneficial. Atypical pig weighing between 145 and about 224 pounds will drink anaverage of 9 liters of water per day. A suggested dose of DFM Bacillusis around 5×10⁹ cfu/pig/day/9 liters of water. A probiotic composition,such as one according to the invention, may provide around 5.5×10⁵cfu/mL to around 6.0×10⁵ cfu/mL. Dosed out over a month, a one literprobiotic composition will provide around 32 mL/day and provide a sporecount of around 6.0×10⁵ cfu/mL or a total of 5.4×10⁹ spores/day—theamount needed per pig. Therefore, a one liter supply will last aroundone month for a single pig in this weight range. Smaller pigs orchickens or other types of animals would typically require smaller dosesof probiotics, which would make a one liter supply last longer or besufficient to dose a larger number of animals. Various factors may alterthese numbers, which are intended to be exemplary and not limiting. Forexample, when dispensed into a water trough with flowing water, thewater flow-rate may also impact the dose that reaches each animaldrinking from the trough. As animals grow, the desirable dose ofprobiotics will increase. Those of ordinary skill in the art willunderstand how to determine the desirable dose, and how to adjust theparameters of the systems of the invention in order to achieve thosedoses, so as to deliver an effective amount of probiotic composition tothe animal or plant consuming the probiotic.

Although primarily described herein with respect to animal watering andfeeding stations, the systems of the invention may also be used todeliver probiotics to plants by delivery to a planter or the soil arounda plant, water tank or cistern, or to aquatic species, such as in a pondor fish tank. The systems of the invention are designed to be easilyprogrammed and re-programmed at the point of consumption to adjust theamount of probiotic dispensed to achieve the desired doses of probioticsbased on the variables present.

Generally, overdosing is not problematic for the animal or plantinvolved, but may result in wasting the probiotic composition, whichincreases the costs involved. Additionally, when used with the probioticcompositions of the invention, the bacteria should be able to survivefor several hours once dispensed from the system, and may even germinatein the drinking water or pond or fish tank, if that is the point ofconsumption to which the probiotic is dispensed. While it is an objectof the invention to provide a system and method that will efficientlymaximize delivery of the probiotic composition to the intended animalsor plants, rather than the compositions being wasted because they arenot consumed before the bacteria are no longer viable, the timing ofdelivery and dosage amounts need not be precise.

Most preferably, systems 10, 110, and 210 are used to dispense probioticand/or synbiotic compositions to animal drinking water in conjunctionwith acidified drinking water. As mentioned above, probioticcompositions according to the invention may include additionalacidifiers that may be used to acidify water delivered to animals.Because the size of the probiotic containers are typically relativelysmall, using the container of probiotic composition to acidify thedrinking water is feasible only in small scale situations involving asingle, smaller sized trough or drinking station. When a larger scalewatering system is used, it is preferred to have a separateacidification system to be used in conjunction with system 10, 110, or210. Various acidizing products are commercially available, such as VevoVital, Acid LAC, Seiko-pH, Lupro-COD NA, and Amasil NA. Generally, theyare used with a dosing/injection system, such as one commerciallyavailable from Dosatron.

Although commercially available acidifiers (which may contain a singleacid or blend of acids or salts of acids) may be used in conjunctionwith system 10, 110, or 210, certain acids or salts of acids arepreferred to be used to acidify the drinking water based on theirantimicrobial activity. For example, acetic acid inhibits growth of E.coli and Salmonella; propionic acid and sorbic acid are antifungal(yeasts, molds) and have anti-bacterial activity with respect to E. coli(including ETEC), Coliforms, and Salmonella; lactic acid also has highanti-bacterial activity with respect to E. coli (including ETEC),Coliforms, and Salmonella, however it can be metabolized by many yeastsand molds; fumaric acid has anti-bacterial activity for E. coli(including ETEC), Coliforms, and Clostridia; citric and benzoic acidshave anti-bacterial activity for E. coli (including ETEC) and Coliforms.Many common salts of these acids, such as calcium formate, calciumpropionate, potassium diformate, potassium sorbate, sodium butyrate,sodium benzoate, and sodium formate, similarly have antimicrobialactivity. Most preferably, the acids selected to acidify the drinkingwater have a pH value lower than the pKa value so that the undissociatedform will be dominate. The undissociated form is desirable because it isable to penetrate the cell wall of the pathogenic bacteria, withoutnegatively impacting the beneficial bacteria in the probioticcomposition. Many of these acidifiers are included in the preferred listof preservatives or acidifiers used with probiotic compositionsaccording to the invention. As will be understood by those of ordinaryskill in the art, different dosing rates for acidifiers will be used,depending on the number, type, age, and size of animals, seasonal andenvironmental conditions (as animals will usually consume more waterduring periods of elevated temperatures and during daylight or simulateddaylight hours). Most preferably, the water in the water system istested to determine its pH before adding any acidifier and the amount ofacidifier added is adjusted based on that base measurement. This avoidsadding too much (which may be harmful to the animals and water systemequipment) or too little acidifier (which eliminates the benefits toeither or both the water system and animals). It is preferred thatsufficient acids or salts of acids be added to the drinking water toachieve a pH in the range of about 4.5 to 6.5, most preferably betweenabout 4.5 to 5.0.

An additional benefit of DFM using compositions according to theinvention is that many of the Bacillus species, will survive through theintestinal tract and remain viable in feces as either spores orvegetative forms. Having these beneficial bacteria in the feces aids inreducing odors associated with the animal waste products. Althoughtreatment compositions containing bacteria may be directly applied toanimal waste, such as manure piles, housing and bedding to reduce odors,a problem frequently encountered is that it may be difficult toadequately and evenly distribute the bacterial treatment over thesurfaces having substances that produce the odors, and particularly todistribute the bacterial treatment through a pile of manure. Having thetreatment bacteria in the feces of the animal through a DFM applicationaids in evenly distributing the beneficial bacteria throughout the fecesand throughout manure piles or storage facilities.

Those of ordinary skill in the art will also appreciate upon readingthis specification, that modifications and alterations to the probioticcompositions and methodology and system for delivery of probioticcompositions may be made within the scope of the invention and it isintended that the scope of the invention disclosed herein be limitedonly by the broadest interpretation of the appended claims to which theinventors are legally entitled.

We claim:
 1. A probiotic composition for treating animals, plants, oranimal bedding, the composition comprising: one or more Bacillus speciesin spore form; one or more acids or salts of acids; and a thickener. 2.The probiotic composition according to claim 1 wherein the bacterialspecies comprises one or more of: Bacillus pumilus, Bacilluslicheniformis, Bacillus amylophilus, Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus clausii, Bacillus firmus, Bacillusmegaterium, Bacillus mesentericus, Bacillus subtilis var. natto, orBacillus toyonensis.
 3. The probiotic composition according to claim 2wherein the composition has a pH less than or equal to 6.5.
 4. Theprobiotic composition of claim 3 wherein the acids or salts of acids areone or more of acetic acid, citric acid, fumaric acid, propionic acid,sodium propionate, calcium propionate, formic acid, sodium formate,benzoic acid, sodium benzoate, sorbic acid, potassium sorbate, orcalcium sorbate.
 5. The probiotic composition of claim 4 wherein thethickener is one or more of xanthan gum, acacia gum, locust bean gum,guar gum or gum arabic.
 6. The probiotic composition of claim 1comprising bacteria spore counts of around 1.0×10⁸ to 3.0×10⁸ cfu/mL ofcomposition, about 0.1 to 5.0% by weight thickener, and 0.05 to 0.5% byweight total of one or more acids or salts of acids.
 7. The probioticcomposition of claim 6 further comprising water.
 8. The probioticcomposition of claim 7 wherein the bacteria spores are mixed with saltto form a dry powder blend prior to being added to the other ingredientsof the composition to form an aqueous composition and wherein thecomposition comprises around 0.05 to 0.3% by weight of the dry powderblend.
 9. The probiotic composition of claim 8 wherein the bacteriaspores are suspended and evenly distributed in the aqueous composition.10. The probiotic composition of claim 6 further comprising anadditional 0.1 to 20% by weight total of one or more additional acids orsalts of acids to acidify the composition.
 11. The probiotic compositionof claim 1 further comprising a surfactant.
 12. The probioticcomposition of claim 11 wherein the surfactant is polysorbate
 80. 13.The probiotic composition of claim 6 further comprising a water activityreducer.
 14. The probiotic composition of claim 13 wherein the wateractivity reducer is one or more of sodium chloride, potassium chloride,or a 70% corn syrup solution.
 15. The probiotic composition of claim 14comprising around 0.1-20% by weight total of one or more water activityreducers.
 16. The probiotic composition of claim 3 wherein thecomposition has a pH between about 3.5 and 5.5.
 17. A system fordelivering a treatment composition to a point of consumption for a plantor animal or to animal housing or bedding, the system comprising: acontainer having an initial volume of treatment composition; a tube influid communication with the container to deliver an amount of treatmentcomposition from the container to a point of consumption by the plant oranimal; a controller for controlling the flow of treatment compositionthrough the tube; wherein the container is sealed to preventcontamination of the treatment composition within the container.
 18. Thesystem of claim 17 further comprising a non-contact pump to pump thetreatment composition from the container through the tube.
 19. Thesystem of claim 17 further comprising a valve to start and stop the flowof treatment composition through the tube.
 20. The system of claim 19wherein the treatment composition is delivered by gravity feed.
 21. Thesystem of claim 17 wherein the tube is integrally formed with thecontainer or attachable to the container at one end.
 22. The system ofclaim 17 further comprising a housing to protect the container.
 23. Thesystem of claim 17 wherein the treatment composition comprises: one ormore Bacillus bacterial species in spore form; one or more acids orsalts of acids; and a thickener.
 24. The system of claim 23 wherein thebacterial species comprises one or more of: Bacillus pumilus, Bacilluslicheniformis, Bacillus amylophilus, Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus clausii, Bacillus firmus, Bacillusmegaterium, Bacillus mesentericus, Bacillus subtilis var. natto, orBacillus toyonensis.
 25. The system of claim 24 wherein the treatmentcomposition has a pH less than or equal to 6.5.
 26. The system of claim25 wherein the acids or salts of acids are one or more of acetic acid,citric acid, fumaric acid, propionic acid, sodium propionate, calciumpropionate, formic acid, sodium formate, benzoic acid, sodium benzoate,sorbic acid, potassium sorbate, or calcium sorbate.
 27. The system ofclaim 26 wherein the thickener is one or more of xanthan gum, acaciagum, locust bean gum, guar gum or gum arabic.
 28. The system of claim 23wherein the treatment composition comprises Bacillus species sporecounts of around 1.0×10⁸ to 3.0×10⁸ cfu/mL of composition, about 0.1 to5.0% by weight thickener, and 0.05 to 0.5% by weight total of one ormore acids or salts of acids, and water.
 29. The system of claim 17wherein the point of consumption is animal drinking water and furthercomprising: a second container having an initial volume of one or moreacids or salts of acids; and a dosing mechanism for delivering an amountof the acids or salts of acids from the second container to the drinkingwater to acidify the drinking water.
 30. The system of claim 29 whereinthe acidified drinking water has a pH of between about 4.5 and 6.5. 31.The system of claim 29 wherein acids or salts of acids in the secondcontainer are one or more of acetic acid, propionic acid, sorbic acid,lactic acid, citric acid, benzoic acid, fumaric acid, calcium formate,calcium propionate, potassium diformate, potassium sorbate, sodiumbutyrate, sodium benzoate, or sodium formate.
 32. The system of claim 29wherein the treatment composition comprises: one or more bacterialspecies in spore form; one or more acids or salts of acids; and athickener.
 33. The system of claim 19 wherein the valve is a meteringvalve comprising a piston actuator and wherein the controller is a motorconnected to a timer that actuates the piston to open and close thevalve for a predetermined amount of time to achieve a desired dosage oftreatment composition and cycles opening and closing the valve atpredetermined intervals.
 34. A method for increasing beneficialbacterial populations in the gastrointestinal tracts of animals, themethod comprising: providing drinking water; adding an amount of aprobiotic composition comprising one or more bacteria species to thedrinking water; acidifying the drinking water by adding an amount of oneor more acids or salts of acids to the drinking water; and wherein theprobiotic composition and acids or salts or acids are added prior to orduring a time when an animal will drink the drinking water.
 35. Themethod of claim 34 wherein the probiotic composition is addedcontinuously or periodically and wherein the acids or salts of acids areadded continuously or periodically.
 36. The method of claim 34 whereinthe acidified drinking water has a pH of between about 4.5 and 6.5. 37.The method of claim 36 further comprising measuring the pH of thedrinking water prior to the acidifying step and altering the amount ofacids of salts of acids added based on the pH measurement.
 38. Themethod of claim 34 wherein the acids or salts of acids are one or moreof acetic acid, propionic acid, sorbic acid, lactic acid, citric acid,benzoic acid, fumaric acid, calcium formate, calcium propionate,potassium diformate, potassium sorbate, sodium butyrate, sodiumbenzoate, or sodium formate.
 39. The method of claim 34 wherein theprobiotic composition comprises: one or more bacterial species; one ormore acids or salts of acids; and a thickener.
 40. The method of claim39 wherein the bacterial species comprises one or more of: Bacillusgenus, Bacteroides genus, Bifidobacterium genus, Pediococcus genus,Enterococcus genus, Lactobacillus genus, Propionibacterium genus,Leuconostoc mesenteroides, or Megasphaera elsdennii
 41. The method ofclaim 40 wherein the bacterial species comprises one or more of Bacilluspumilus, Bacillus licheniformis, Bacillus amylophilus, Bacillussubtilis, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus firmus,Bacillus megaterium, Bacillus mesentericus, Bacillus subtilis var.natto, or Bacillus toyonensis Bacteroides ruminocola, Bacteroidesruminocola, Bacterioides suis, Bifidobacterium adolescentis,Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacteriuminfantis, Bifidobacterium longum, Bifidobacterium thermophilum,Pediococcus acidilacticii, Pediococcus cerevisiae, Pediococcuspentosaceus, Enterococcus cremoris, Enterococcus diacetylactis,Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis,Enterococcus thermophilus, Lactobacillus delbruekii, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillusbuchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillusfarciminis, Lactobacillus cellobiosus, Lactobacillus curvatus,Propionibacterium acidipropionici, or Propionibacterium freudenreichii,Propionibacterium shermanii
 42. The method of claim 39 wherein thebacterial species are one or more of the Bacillus genus in spore form.43. The method of claim 42 wherein the Bacillus species is one or moreof Bacillus pumilus, Bacillus licheniformis, Bacillus amylophilus,Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus clausii,Bacillus firmus, Bacillus megaterium, Bacillus mesentericus, Bacillussubtilis var. natto, or Bacillus toyonensis.
 44. The method of claim 42wherein the probiotic composition has a pH less than or equal to 6.5.45. The method of claim 44 wherein the acids or salts of acids in theprobiotic composition are one or more of acetic acid, citric acid,fumaric acid, propionic acid, sodium propionate, calcium propionate,formic acid, sodium formate, benzoic acid, sodium benzoate, sorbic acid,potassium sorbate, or calcium sorbate.
 46. The method of claim 45wherein the thickener is one or more of xanthan gum, acacia gum, locustbean gum, guar gum or gum arabic.
 47. The method of claim 46 wherein theprobiotic composition comprises bacteria spore counts of around 1.0×10⁸to 3.0×10⁸ cfu/mL of composition, about 0.1 to 5.0% by weight thickener,and 0.05 to 0.5% by weight total of one or more acids or salts of acids.48. The method of claim 47 wherein the probiotic composition furthercomprises around 0.1 to 20% by weight total of one or more wateractivity reducers.
 49. The method of claim 48 wherein the water activityreducer is one or more of sodium chloride, potassium chloride, or a 70%corn syrup solution.
 50. The method of claim 34 wherein the probioticcomposition is added to the drinking water using a dosing and deliverysystem comprising: a container having an initial volume of probioticcomposition; a tube in fluid communication with the container to deliveran amount of the probiotic composition from the container to thedrinking water; a controller for controlling the flow of the probioticcomposition through the tube; and wherein the container is sealed toprevent contamination of the probiotic composition within the container.51. The method of claim 50 wherein the controller comprises a motor,wherein the valve comprises a piston, and wherein the motor comprises aninternal timer that actuates the piston to open and close the valve fora predetermined amount of time to achieve a desired dosage of probioticcomposition and cycles opening and closing the valve at predeterminedintervals.
 52. A method for controlling odors associated with animalwaste products, the method comprising: providing drinking water for ananimal; acidifying the drinking water by adding an amount of one or moreacids or salts of acids to the drinking water; and adding an amount of aprobiotic composition comprising one or more bacteria species in sporeform to the drinking water such that at least some of the bacteriaspecies survive through the animal's gastrointestinal tract and arepresent in spore or vegetative form in the animal's feces; and whereinthe probiotic composition and acids or salts or acids are added prior toor during a time when an animal will drink the drinking water and thebacteria in the feces reduce odor causing compounds.
 53. The method ofclaim 52 wherein the bacteria species is one or more Bacillus species.